The invention relates to the electrophoresis process wherein biological cells, colloidal particles, or macromolecules with a net electrical charge migrate and separate in a solution under the influence of an electric field and, more particularly, to a device for carrying out such a process.
The conventional continuous flow electrophoresis device is made predominantly of transparent materials, usually glass and plexiglas to facilitate observation of the separation process. The sample material being separated usually consists of a mixture of biological cells dispersed in a weak electrolyte or buffer. The chamber configuration is rectangular with electrodes on each side running down the long side of the chamber. The sample cells are inserted into the center of a flowing, thin curtain of buffer through a tube of narrower cross-section than the buffer curtain. An electrical field across the width of the chamber causes the injected sample filament to be separated into fraction streams through differential migration of the different cell species as the buffer curtain moves from one end of the chamber to the other. The electrodes are usually contained in electrode chambers which are partitioned from the separation chamber by a flat segment of dialysis membrane material. An array of collection tubes located along the end of the chamber collects the fraction bands along with all flowing buffer. The system provides for a continuous insertion, separation, and collection of injected sample and buffer.
In order to obtain optimum performance in an electrophoresis device, the fluid flow must remain essentially rectilinear even under the imposition of high voltage gradients. Present practice is to use either thin chambers and high voltage gradients or thicker chambers and very low voltage gradients. While these techniques do lower the temperature gradient in the chamber cross section thereby suppressing buoyancy induced disturbances, a large penalty is incurred in system performance. Also, current design practice is to build the chambers from transparent materials in order to observe the ongoing separation process. Transparent materials are difficult to sterilize and in general have poor thermal conductivity.
One attempt to solve this problem is the device shown in U.S. Pat. No. 3,519,549 wherein one side of the separation chamber is closed off by an electrically non-conductive plate, which is preferably transparent such as glass however since the plate is electrically non-conductive, it is also a poor thermal conductor. The other side of the separation chamber is closed off by a heat distribution plate having good thermal conductivity. However, thermal asymmetry is introduced into the chamber by providing good heat removal on one side and poor heat removal on the other. This uneven heat removal gives an asymmetrical thermal gradient in the chamber and adverse flow circulation can be set up by the unstable condition of warm fluid being at one side than at the other.
The buffer curtain that flows inside the rectangular electrophoresis chamber must be very thin, generally 0.5 to 1.5 mm, mainly because neither the fluid nor transparent chamber efficiently conduct the Joule heat out of the buffer. Since the buffer curtain is the "carrier" of the sample, the injected sample stream must be thinner than the buffer curtain. A buffer cross-flow circulation, perpendicular to the sample insertion is caused by interior walls of the chamber being charged (a phenomenon called electro-osmosis) and sample injected into the buffer near to these charged walls will be displaced by this electro-osmotic flow. The combination of electro-osmotic cross flow and Poiseuille flow of the buffer through the chamber require that the sample be inserted as a very narrow stream in the center of the buffer curtain. This, however, limits the amount of sample that can be processed in a given time interval.
The electrode chambers in conventional continuous flow electrophoresis devices are separated from the main electrophoresis chamber by dialysis or ion-exchange membranes. These membranes allow passage of the ions from electrodes to the electrophoresis chamber and inhibit passage of bubbles formed by electrolysis at the electrodes. During electrophoresis, the electrode buffer pH and conductivity drops near the cathode (negative electrode) and increases near the anode (positive electrode). These changes in ion concentration extend into the electrophoresis chamber and changes in pH of one unit and conductivity shifts of a factor of 10 have been measured in the conventional chambers near the membrane. This variation in buffer conductivity plus the normal heating of the membrane causes thermal convection and disturbance to the electrophoresis process.
The collection system for conventional continuous flow electrophoresis chambers consists of hollow tubes, either polymeric or stainless steel, that extend across the width of the chamber and partition the flow. Each element of the buffer curtain is carried by the tubes to individual glass containers that collect the buffer and separated sample. The hollow tubes necessarily have narrow bores comparable to the sample insertion tubes to give the required high resolution of separation. These narrow tubes have a propensity to clog periodically with aggregated cells and cell debris and thus disturb the laminar flow back into the chamber. Any change in flow properties of the tubes thus interferes with the consistent separation of the sample. Analysis of results is also difficult with conventional continuous flow electrophoresis systems because the containers must be individually examined to determine where the separated cells are.
Accordingly, an important objective of the present invention is to provide a continuous flow electrophoresis separation device in which the resolution and throughput is increased by utilizing proper thermal control of the chamber to suppress buoyancy induced convection along with other improvements in the sample insertion and collection system, electrode chambers, and inlet and exit flow conditions.